IL-7 Receptor Mutations and Steroid Resistance in Pediatric T cell Acute Lymphoblastic Leukemia: A Genome Sequencing Study
IL-7 Receptor Mutations and Steroid Resistance in Pediatric T cell Acute Lymphoblastic Leukemia: A Genome Sequencing Study
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DOI：
10.1371/journal.pmed.1002200
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发表时间：
2016-12-01
期刊：
影响因子：
15.8
通讯作者：
Meijerink, Jules P. P.
中科院分区：
文献类型：
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作者：
Li, Yunlei;Buijs-Gladdines, Jessica G. C. A. M.;Meijerink, Jules P. P.
研究背景小儿急性淋巴细胞白血病（ALL）是儿童期最常见的恶性肿瘤，也是儿童癌症相关死亡的主要原因。T细胞ALL（T-ALL）约占儿童ALL病例的15%，被认为是一种高危疾病。T-ALL通常与对治疗的抵抗有关，包括类固醇，这是目前治疗ALL的基石;此外，最初的类固醇反应强烈预测生存和治愈。然而，T-ALL患者类固醇耐药的细胞机制知之甚少。在这项研究中，我们结合了各种基因组数据集，以确定候选的遗传机制，类固醇耐药的儿童接受T-ALL treatment.Methods和FindingsWe进行全基因组测序配对治疗前（诊断）和治疗后（缓解）样本13例，和有针对性的外显子组测序的预处理样本从69个额外的T-ALL患者。然后，我们整合了151个突变基因的突变数据和拷贝数数据，并对该整合数据集进行了突变与临床结果和体外药物反应的相关性测试。我们的分析显示，JAK 1和KRAS（编码白细胞介素7受体（IL 7 R）信号通路组分的两个基因）突变与类固醇耐药和不良结局相关。然后，我们对这69名T-ALL患者和另外77名T-ALL患者的JAK 1、KRAS和该途径中的其他基因（包括IL 7 R、JAK 3、NF 1、NRAS和AKT）进行了测序。我们在32%（47/146）的患者中发现了突变，其中大多数患者具有特定的T-ALL亚型（早期胸腺祖细胞ALL或TLX）。基于这些患者的结果和他们在体外测量的泼尼松龙反应性，我们随后证实这些突变与类固醇抵抗和不良结果相关。为了探索这些IL 7 R信号通路基因突变如何导致类固醇抵抗和随后的不良结果，我们使用诱导型慢病毒表达构建体在两种类固醇敏感性T-ALL细胞系（SUPT 1和P12 Ichikawa细胞）中表达野生型和突变型IL 7 R信号分子。我们发现，表达突变型IL 7 R、JAK 1或NRAS，或野生型NRAS或AKT，特异性诱导类固醇耐药，而不影响对长春新碱或L-天冬酰胺酶的敏感性。相反，野生型IL 7 R、JAK 1和JAK 3以及突变型JAK 3和突变型AKT没有影响。然后，我们进行了一项功能研究，以检查类固醇耐药的机制，并发现，而不是改变类固醇受体的能力，激活下游目标，类固醇耐药与MEK-ERK和AKT，IL 7 R信号通路的下游组件的强烈激活，从而诱导一个强大的抗凋亡反应，通过上调MCL 1和BCLXL表达。MEK-ERK和AKT途径也抑制激素诱导细胞死亡的必需分子BIM，并抑制促凋亡BIM的重要调节因子GSK 3B。重要的是，用IL 7 R信号传导抑制剂治疗我们的细胞系恢复了类固醇敏感性。为了解决临床相关性，我们用类固醇单独或与IL 7 R信号传导抑制剂组合处理了从11名患者获得的原代T-ALL细胞;我们发现，包括MEK、AKT、mTOR或双重PI 3 K/mTOR抑制剂强烈增加了类固醇诱导的细胞死亡。因此，将这些抑制剂与类固醇治疗相结合可能会增强ALL患者的类固醇敏感性。我们研究的主要局限性是队列规模较小，因为T-ALL的发病率非常低。结论使用无偏见的测序方法，我们发现IL 7 R信号分子中的特定突变是T-ALL类固醇耐药性的基础。未来的前瞻性临床研究应测试MEK、AKT、mTOR或PI 3 K/mTOR抑制剂恢复或增强类固醇敏感性并改善临床结局的能力。
BackgroundPediatric acute lymphoblastic leukemia (ALL) is the most common childhood cancer and the leading cause of cancer-related mortality in children. T cell ALL (T-ALL) represents about 15% of pediatric ALL cases and is considered a high-risk disease. T-ALL is often associated with resistance to treatment, including steroids, which are currently the cornerstone for treating ALL; moreover, initial steroid response strongly predicts survival and cure. However, the cellular mechanisms underlying steroid resistance in T-ALL patients are poorly understood. In this study, we combined various genomic datasets in order to identify candidate genetic mechanisms underlying steroid resistance in children undergoing T-ALL treatment.Methods and FindingsWe performed whole genome sequencing on paired pre-treatment (diagnostic) and post-treatment (remission) samples from 13 patients, and targeted exome sequencing of pretreatment samples from 69 additional T-ALL patients. We then integrated mutation data with copy number data for 151 mutated genes, and this integrated dataset was tested for associations of mutations with clinical outcomes and in vitro drug response. Our analysis revealed that mutations in JAK1 and KRAS, two genes encoding components of the interleukin 7 receptor (IL7R) signaling pathway, were associated with steroid resistance and poor outcome. We then sequenced JAK1, KRAS, and other genes in this pathway, including IL7R, JAK3, NF1, NRAS, and AKT, in these 69 T-ALL patients and a further 77 T-ALL patients. We identified mutations in 32% (47/146) of patients, the majority of whom had a specific T-ALL subtype (early thymic progenitor ALL or TLX). Based on the outcomes of these patients and their prednisolone responsiveness measured in vitro, we then confirmed that these mutations were associated with both steroid resistance and poor outcome.To explore how these mutations in IL7R signaling pathway genes cause steroid resistance and subsequent poor outcome, we expressed wild-type and mutant IL7R signaling molecules in two steroid-sensitive T-ALL cell lines (SUPT1 and P12 Ichikawa cells) using inducible lentiviral expression constructs. We found that expressing mutant IL7R, JAK1, or NRAS, or wild-type NRAS or AKT, specifically induced steroid resistance without affecting sensitivity to vincristine or L-asparaginase. In contrast, wild-type IL7R, JAK1, and JAK3, as well as mutant JAK3 and mutant AKT, had no effect. We then performed a functional study to examine the mechanisms underlying steroid resistance and found that, rather than changing the steroid receptor's ability to activate downstream targets, steroid resistance was associated with strong activation of MEK-ERK and AKT, downstream components of the IL7R signaling pathway, thereby inducing a robust antiapoptotic response by upregulating MCL1 and BCLXL expression. Both the MEK-ERK and AKT pathways also inactivate BIM, an essential molecule for steroid-induced cell death, and inhibit GSK3B, an important regulator of proapoptotic BIM. Importantly, treating our cell lines with IL7R signaling inhibitors restored steroid sensitivity. To address clinical relevance, we treated primary T-ALL cells obtained from 11 patients with steroids either alone or in combination with IL7R signaling inhibitors; we found that including a MEK, AKT, mTOR, or dual PI3K/mTOR inhibitor strongly increased steroid-induced cell death. Therefore, combining these inhibitors with steroid treatment may enhance steroid sensitivity in patients with ALL. The main limitation of our study was the modest cohort size, owing to the very low incidence of T-ALL.ConclusionsUsing an unbiased sequencing approach, we found that specific mutations in IL7R signaling molecules underlie steroid resistance in T-ALL. Future prospective clinical studies should test the ability of inhibitors of MEK, AKT, mTOR, or PI3K/mTOR to restore or enhance steroid sensitivity and improve clinical outcome.